We determined the membrane acceptor site for C3b deposited on epimastigotes (Epi) of Trypanosoma cruzi by the alternative pathway (AP). Epi (Tulahuen strain) were surface-iodinated with Iodogen, then incubated in 50% C8 deficient serum. Washed parasites were solubilized in detergent and the lysate was applied to an anti-C3 column (A:C3). 15.6(plus and minus)1.6% of applied 125I Epi protein bound specifically to A:C3 with .3% binding in controls. Bound 125I Epi protein was eluted from A:C3 and analyzed by SDS-PAGE autoradiography. Non-serum incubated Epi showed 9 major iodinated bands ranging from 20 kd to 208 kd. Samples eluted from A:C3 with SDS showed a single diffuse band at 250-300kd, suggesting covalent attachment of C3b to 125I Epi protein. Samples eluted from A:C3 with NH2OH revealed a single major band at 72kd, suggesting that C3b attached almost exclusively to the 72 kd glycoprotein by a NH2OH susceptible ester bond. An antiserum was prepared from lysates of serum treated Epi affinity purified on A:C3. This antiserum immunprecipitated a single componenet of 72kd from surface iodinated Epi. These results are the first to evaluate the acceptor site for C3 deposition on a parasite, and show that C3b deposited by the AP binds preferentially to the 72kd glycoprotein, the major developmentally regulated surface antigen of Epi.